ES2305534T3 - MESOPOROUS GLASS AS NUCLEANT FOR MOLECULAR CRSTALIZATION. - Google Patents

Abstract

A method of facilitating the crystallisation of a macromolecule comprising the step of adding a mesoporous glass to a crystallisation sample wherein the mesoporous glass comprises pores having diameters between 4 nm and 100 nm and has a surface area of at least 50 m<SUP>2</SUP>/g. A method of facilitating the crystallisation of a macromolecule comprising the step of adding to a crystallisation sample a mesoporous glass of the composition Si0<SUB>2</SUB>; CaO-P<SUB>2</SUB>0<SUB>5</SUB>-SiO<SUB>2 </SUB>or Na<SUB>2</SUB>0-CaO-P<SUB>2</SUB>0<SUB>5</SUB>-SiO<SUB>2</SUB>, wherein each of the Ca, P, Si or Na atoms within the compositions may be substituted with a suitable atom chosen from B, Al, Ti, Mg, or K, and, optionally, the composition may also include heavy elements to enhance X-ray diffraction contrast such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta or other atom with an atomic number over 20.

Description

Mesoporous glass as a nucleant for macromolecular crystallization.

The present invention relates to the use of a new nucleant in the crystallization of macromolecules and methods of crystallizing macromolecules such as proteins, using The new nucleant.

To date, the nucleation of glass has been facilitated mainly by planting, epitaxy, loaded surfaces or mechanical means (Stura, EA In Protein crystallization: techniques, strategies, and tips. (Ed Bergfors, TM) (International University Line, La Jolla; 1999); McPherson, A. & Shlichta, P. Science 239, 385-387 (1988); Sanjoh, A. & Tsukihara, TJ Cryst. Growth 196, 691-702 (1999); Sanjoh, A., Tsukihara, T .and Gorti, SJ Crystal Growth232 618-628 (2001); Visuri, K. et al . Bio / Technology 8,547-549 (1990)). Glass nucleation is the first necessary step in the crystallization process and its influence in this process is decisive. Consequently, the ability to control it is of crucial importance in crystallization experiments. Nucleation presents a free energy barrier that must be overcome in a particular way, different from the supersaturation conditions that subsequently make glass growth an energy-efficient process (Feher, G. & Kam, Z. Methods Enzymol. 114, 77 -112 (1985)). The formation of nuclei in the bulk of a solution is a stochastic process in which protein molecules interact to form an aggregate of critical size. Any environment that favors a higher local concentration of macromolecules will provide a potential nucleation point and may reduce the energy barrier to nucleation.

Pilot projects of structural genomics show a success rate of around 10% in passing replication  to structure. It has been discovered that the stage that limits this index is the production of glasses suitable for crystallography with X-rays (eg the Human Proteome Structural Genomics pilot project; Brookhaven National Laboratory, The Rockefeller University and Albert Einstein College of Medicine: http://proteome.bnl.gov/progress.html). It is currently It is essential to find methods that help overcome this obstacle.

The ultimate goal would be to find a nucleant "universal" that could favor the crystallization of a wide range of proteins under a wide range of conditions. Previously studies have been undertaken in order to discover nucleants introducing candidate substances in a controlled manner in crystallization experiments (McPherson and Schlichta Science 239, 385-387 (1988); Chayen, N.E., Radcliffe, J.W. & Blow, D.M. Protein Sci. 2, 113-118 (1993); Blow, D.M., Chayen, N.E., Lloyd, L.F. & Saridakis, E. Protein Sci. 3, 1638-1643 (1994); Malkin, A.J., Cheung, J and McPherson, A. (1993) J. Crystal Growth 126 544-554 (in relation to "Mcpherson Beads" -latex beads / particles of latex "McPherson Particles")). Some have had success with individual proteins but for now none has turned out to be general use Various other attempts to induce nucleation in irregular or rough surfaces, or compositional surfaces especially, they have also proved ineffective in general (Chayen, N.E. & Saridakis, E. J. Cryst. Growth 232, 262-264 (2001).

Sakamoto et al . Nature 408, 449-453 (2000) and Dusastre Nature 408: 417 (2000) describe methods for characterizing mesoporous materials and disclose the structures of some mesoporous materials. It is suggested that these are useful in several areas of chemistry such as catalysis and molecular filtration, but their use for glass nucleation is not alluded to.

We have discovered that mesoporous glasses with particular characteristics are useful as nucleants in the crystallization. Mesoporous glasses are considered to have pores that trap macromolecules, for example protein molecules, and propel them to nuclear and form glass.

A first aspect of the invention reveals a method to facilitate the crystallization of a macromolecule that comprises the step of adding mesoporous glass to the sample of crystallization, mesoporous glass that contains pores with diameters between 4 nm and 100 nm and has a surface area of at minus 50 m2 / g.

In certain embodiments, the glass mesoporous contains pores with diameters between 4 nm and 70 or 80 nm, 4 nm and 40 nm, or 7 nm and 40 nm. In the embodiment described in the Example 2, where the mesoporous glass has pores with diameters between 7 and 40 nm, the modal pore size is 11.2 nm and the average pore size It is 16 nm.

Pore size distributions are calculated using the standard technique in the field, i.e. from isothermal gas absorption and desorption data (for example, N_ {2} or He) using the BJH method (Barrett EP, Joyney LG, Halenda PP Jof Am. Chem. Soc. 1951; 73: 373-380), as described in Examples 1 and 2. Using this method, less than 10%, 5% or 2% (by number) of the pores leave the range of specified size, for example, a range between 4 nm and 100 nm Pore size can show a wide distribution Gaussin in the interval in question. It is considered that an interval of pore sizes provides good properties of nucleation; a narrow size distribution of pore, in which for example more than 80, 85 or 90% of the pores have a pore size within the range between X nm and (X + 10 nm) or (X + 5 nm), where X is in a size range specific, it will not provide nucleation properties equal to good.

As used herein, the term "mesoporous glass" includes a glass with pores without order and irregular sizes whose diameters can vary between a few nanometers and tens or hundreds of nanometers. The glass can be the fusion of one or more silicon, boron and phosphorus oxides, with one or more sodium, magnesium, calcium or potassium oxides, and optionally sodium or calcium carbonate. Alternatively, and preferably, mesoporous glass with pores of unordered and irregular sizes whose diameters can range between a few nanometers and tens or hundreds of nanometers, can be manufactured by creating a gel using SiO2 alkoxid precursors as tetramethylortosilicate (TMOS ) or tetraethylorthosilicate (TEOS) hydrolyzed with H2O and a catalyst (such as HNO3 or HF) to make silica glass (SiO2) mesoporous or hydrolyzed with H2O and a combination catalyst with P 2 O 5 alkoxid precursors, such as triethoxyl orthophosphate (PET, OP (OC 2 H 5) 3), or CaO nitrate precursors, such as Ca (NO 3)) 2, or Na 2 O strokes, as sodium citrate. Examples include SiO 2, CaO-P 2 O 5 -SiO 2 or Na 2 O-CaOP 2 O 5 -SiO 2. Mesopores appear in the sun-gel derived glass when the three-dimensional network is being established during gelation and are maintained following a controlled drying technique well known to those skilled in the art and described in Hench (1997) Curr Opin Solid State Mater Science 2, 604-610, Li et al (1991) J Appl Biomaterials 2, 231-239, Hench and West (1990) Chem Rev 90, 33-72.

The pores of the mesoporous glass useful in the present invention have diameters between 4 nm and 100 nm (or of other intervals in some embodiments, as noted above), determined using a method of analysis by conventional gas absorption, as described in Coleman & Hench (2000) A gel-derived mesoporous silica reference material for surface analysis by gas surprise 1: Textural features Ceramics International 26, 171-178. By example, pore sizes can be calculated from the data of desorption of a standard gas (for example, N2) using the BJH method (cf. Example 2 and [5]). Mesoporous glass can Hold a heterogeneous sample of pore sizes. The material may have open cylindrical pores, determined by isotherms gas absorption / desorption (for example, N2), as is known by those skilled in the art and as described in Example 2. The hysteresis loop can be of type H1 (Sing KSW, Everett DH, Haul RAW. Pure and Appl. Chem. 1985; 57: 603-619), the which indicates that the material mainly contains pores open and interconnected cylindrical that are not closed in the surface.

Mesoporous glass for use in the method of invention may have a surface area of at least 250 m2 / g, 200 m2 / g, 150 m2 / g, 100 m2 / g, or at least 50 m 2 / g. The surface area will preferably be less than 300 m 2 / g. In one embodiment, the surface area is between 150 m 2 / g and 100 m 2 / g. The surface area of the glass mesoporous is a measure of both pore size and number of pores, and can be increased or reduced during the procedure sol-gel, as experts in the technique and as described in Hench, L.L. (1998) Sol-Gel Silicas, Hayes Publishing Co, New York, and in Iler, R.K. (1979) The Chemistry of Silica, J Wiley & Sons, New York The surface area of mesoporous glass can calculated using a gas absorption isotherm (for example N2), as described later in Example 2, or any other equivalent surface analysis method.

Mesoporous glasses that may be of particular utility in the method of the invention are disclosed in Example 1 and in Cook et al (2000) Bioceramics, 13, 625-628; Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8; Pereira & Hench (1996) J Sol-gel Sci Tech 7, 59-68; Li et al (1992) Chem Proc Adv Mater 56, 627-633; Lenza et al (2002) J Mater Sci, Mat in Med 13, 837-892; Orefice et al (2001) J Biomed Mater Res 55, 460-467.

It will be understood that when we use the term "macromolecule" we are including any higher molecule at 1 kDa. Preferably the macromolecule will be a macromolecule biological as a nucleic acid, and more preferably the Macromolecule will be a polypeptide. Preferably the polypeptide it will comprise at least 10, 20 or 50 amino acids, and more preferably at least 75, 100, 200, 500 or 1000 amino acids.

A second aspect of the invention provides a method to facilitate the crystallization of a macromolecule that it comprises the step of adding a glass to the crystallization sample mesoporous of the composition SiO2, CaO-P 2 O 5 -SiO 2 or Na 2 O-CaO-P 2 O 5 -SiO 2, where each of the atoms of Ca, P, Si or Na within the compositions can be substituted by an appropriate atom chosen between B, Al, Ti, Mg or K, and, optionally, the composition may also include heavy elements to accentuate the contrast of X-ray diffraction, such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta or another atom with an atomic number greater than 20.

In a preferred embodiment of the second aspect of the invention, mesoporous glass will have the composition SiO_ {2}, CaO-P 2 O 5 -SiO 2 or Na 2 O-CaO-P 2 O 5 -SiO 2.

CaO-P2O5 -SiO2 or Na2O-CaO-P2O5 -SiO2 are bioactive mesoporous glasses that have been considered useful for bone regeneration (Saravanapavan and Hench (2001) J Biomed Mater Res. 54, 608-18; Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8 Cook et al (2000) Bioceramics, 13, 625-628). The Na 2 O-CaO-P 2 O 5 -SiO 2 composition is also bioactive and is described in detail in Hench & West (1996) Life Chem Prep 13, 187-241. The bioactive bone binding response of the mesoporous glass of SiO2 has been described by Hench & Walker (1979) in US Pat. No. 4171544.

Example 1 demonstrates the fitness of the glasses mesoporous as nucleating to facilitate the crystallization of macromolecules

By "substituted by an atom appropriate "we mean that the atom chosen from the selection that is offered has the same valence as the substituted atom, e.g. ex. Mg for Ca, K for Na; alternatively, valence atoms multiple as P can be substituted for Si in the networks of the glass.

It is preferable that the mesoporo glass used in the second aspect of the invention comprise pores with diameters between 200 nm, 150 nm or 100 nm and 2, 4, 7, 10, or 20 nm. Even more preferably, the pore diameters and their distribution will be as indicated above in relation to the first aspect of the invention. Thus, in embodiments of the second aspect of the invention the mesoporous glass that is used in the method will comprise pores with diameters between 4 nm and 100 nm or 7 nm and 40 nm, measured using conventional methods, as described, for example, in Example 2.

In relation to the first or second aspect of the invention, the distribution of pore dimensions of the mesoporous glass that is used in the method of the invention has a standard deviation of at least 5 nm, 10 nm or 15 nm.

Usually the mesoporous glass that is used in the second aspect of the invention it is distinguished by a uneven distribution of pore sizes within the same. Therefore, it is preferable that the pores of the mesoporous glass do not have a uniform size and have high variability in As for the size of the pores.

In a further embodiment of the methods of The invention, mesopore glass has interconnected pores. By example, the pores may include open cylindrical pores, such as It is described in Example 2.

As used herein, the term "pores interconnected "includes a mesoporous glass in which at least some pores are interconnected so that the contents of a Pore or cavity are accessible to one or more pores or cavities adjacent or connected and, like open pores, intersect with the surface.

Generally, the interconnected nature of the pores is shared by at least 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70% of the pores of the mesoporous glass. Preferably, the mesoporous glass has an H1 pore classification as described in Sing et al (1985) Pure and Appl Chem 57, 603-619, ie the mesoporous glass primarily contains open cylindrical pores that are interconnected.

The porosity of CaO-P2O5 -SiO2 and Na2O-CaO-P2O5 -SiO2 can be modified using a foaming technique. For example, the unfoamed CaO-P 2 O 5 -SiO 2 has a surface area of 146.4 m2 / g while a CaO-P 2 O 5 - Low foamed SiO 2 has a surface area of 239.6 m2 / g (Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8). Foaming also alters the average pore size and the three-dimensional nature of the pores and generates very large pores of 100 µm or greater in addition to the mesopores. Thus, the mesoporous glass of composition CaO-P2O5 -SiO2 or Na2O-CaO-P2O5 -SiO2 can be manufactured using the techniques described in Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8 or as described in Pereira & Hench (1996) J Sol-Gel Sci and Tech 7, 59-68 or Li et al (1992) Chem Proc Adv Mater 56, 627-633, in order to generate a porous substance having a surface area suitable for use in the invention, with average pore size dimensions appropriate for use in the invention, and a variability in the average pore size dimensions and with an interconnected pores suitable for use in the invention.

In the crystallization of macromolecules, it has been previously experimented with materials such as Sephadex ™ particles, zeolites and aluminum powders, but these have proved ineffective as commonly used nucleating agents (Chayen, NE & Saridakis, EJ Cryst. Growth 232 , 262-264 (2001)). The Sephadex ™ consists of a network produced by crosslinking substituent material. This crosslink creates pores effectively, the size of which is determined by the degree or type of crosslink. It should be noted that Sephadex ™ and sand are not included within the present definition of "mesoporous glass". One difference between the porous material useful in the present invention and the Sephadex ™ is that the pores of each given type of Sephadex beads have a quasi- uniform size. As said before, the mesoporous glass useful in the present invention comprises pores of varying sizes, as explained above; The material has an average pore diameter generated by a considerably wide Gaussian pore size distribution. Hence, the distribution of pore dimension in the porous material is wider than that of crosslinked dextrins.

It should be noted that the appropriate average size for a given application it can depend on the size of the macromolecule to be crystallized. For example, sets larger macromolecular (i.e. with a size larger than 5 nm or 10 nm or 15 nm Stokes radius) such as viral capsids, etc. may require larger pores (i.e. pores with medium size greater than 10 nm or 20 nm or 50 nm in any plane) than size of pore required by the smallest macromolecular assemblies of the methods of the invention, such as with a radius of Stokes of 5 nm or less.

In a preferred embodiment of the methods of the invention, the crystallization of the macromolecule is induced in a critical level of supersaturation lower than that obtained when Mesoporous glass is not added to the sample.

In a further preferred embodiment or alternative, the conditions of the crystallization sample they include supersaturation states that are favorable for vitreous growth, but inadequate or insufficient for spontaneous nucleation. Such conditions are a means of maximizing the chances of getting glasses during the initial selection of the crystallization conditions and provide a means to cultivate glasses in metastable states in which growth slower and the absence of excess secondary nucleation they often provide a culture of better diffraction glasses and bigger.

Determine what level of supersaturation is inadequate or insufficient for spontaneous nucleation is something well known in the art of crystallization. The level of supersaturation can be determined by installing filters that cover a range of conditions between the conditions that produce spontaneously glass / microcrystals.

Preferably the invention provides a method to facilitate the crystallization of a macromolecule according to the first or second aspect of the invention comprising the step of breaking the mesoporous glass into pieces of dimensions submillimeter before adding mesoporous glass to the sample of crystallization.

Preferably, the mesoporous glass is de novo prepared in pieces of submillimeter dimensions deforming the gel network and stirring at the same time.

Preferably, the mesoporous glass should be converted into pieces or fragments that are not larger than 200 µm, 150 µm or 100 micrometers in any dimension, more preferably not larger than 75 µm or 50 µm, and even more preferably they are not larger than 25 µm in any dimension. More preferably, no dimension of the pieces will be greater than 10 micrometer. Advantageously, the mesoporous glass parts resemble a fine powder. The conversion of mesoporous glass into fragments can be achieved using a diamond cutter or
of SiC.

By "fracturing" we mean that the Initial material is converted into fragments or smaller pieces. It can be fractured by any suitable technique, including cutting with a scalpel or mechanical means (using by example a diamond cutter) or break the large piece in Small pieces using tweezers. Usually glass mesoporous useful in the present invention cannot be broken manually in small enough parts. The surface is mechanically fragile, so it is preferable to polish or crush with reduced loads (for example with vibration). It should be noted that for be useful as nucleants in crystallization, fragments or pieces have to be such that the network of pores inside the Mesoporous glass is exposed. In other words, the pores must be accessible for molecules with a size appropriate (i.e., those that are smaller than the size of the pores) when these molecules come into contact with them. In Consequently, fragments or pieces of mesoporous glass are not they will close outwardly in any way by the procedure of fracturing

The fracturing method can be manual or mechanical or be done with a motor machine.

A third aspect of the invention is a method. to determine the structure of a macromolecule comprising the steps of (i) crystallizing the macromolecule in the presence of a mesoporous glass as defined in relation to the first and the second aspect of the invention, and (ii) analyze the structure of the glass produced in step (i).

The porous materials useful or preferred in the third aspect of the invention are those that have been defined in detail previously. Preferably, the porous material It is produced as described above. The material can be combined in any order with the sample components of crystallization. Preferably, the material will be added before Let the nucleation or growth of crystallization begin.

Nucleation can be detected by any suitable means; either directly, for example, using a microscope, or indirectly, for example by determining the characteristics of light scattering as described in Rosenberger et al. J. Cryst. Growth 129: 1-12 (1993); Saridakis et al Acta Cryst D 58, 1597-1600 (2002).

The methods of glass structure analysis of a glass are well known in the art and are described in detail in Drenth, J. Principles of protein X-ray crystallography Springer-Verlag, New York, 1994

A fourth aspect of the invention is a camera appropriate to crystallize a macromolecule where said chamber it comprises (at least in part) a mesoporous glass as defined in relation to the first and second aspects of the invention. The chamber can be manufactured with mesoporous glass (at least partially; for example in the form of graft), or glass mesoporous may constitute a coating of the chamber (i.e. in a part of the chamber that is not made of mesoporous glass). Should Note that the mesoporous glass will be in a part of the chamber in contact with the crystallization sample, when less at the beginning of the crystallization test.

With "appropriate camera for crystallization "we include a camera on a plate crystallization or multiwell crystallization plate, or a chamber individual, as those skilled in the art will know. By for example, the coverslip of which a drop is suspended in the "pendant drop" crystallization (or in which a drop can perch, in a crystallization of "drop drop") can manufactured or coated with mesoporous glass.

The chambers can be manufactured or coated with mesoporous glass by a procedure similar to that described in Roethen et al (2002) Biomaterials 23, 3871-3878. The coating can be a continuous coating or a coating with mesoporous glass particles.

A further aspect of the invention provides a fiber, mesh or film comprising a mesoporous glass such as is defined in relation to the first and second aspects of the invention. A further aspect of the invention provides the use of said fiber, mesh or film as a nucleant of the crystallization, for example in the method of the invention. The fiber it can be a fiber made of a different material that is coated with a mesoporous glass (as well as a continuous layer, well with mesoporous glass particles). The glasses grow on the fiber, which can be extracted slowly by solution and then allow continuous growth. Thus, the fiber for nucleating is able to be used in a continuous procedure.

The coating can be performed using, for example, a method similar to that described by Stamboulin et al (2002) Adv Eng Met 105-109 and Roether et al (2002) Biomaterials 23, 3871-3878, but not limited thereto.

A fifth aspect of the invention provides a use in the crystallization of a macromolecule of a glass mesoporous as defined in relation to the first or the second aspect of the invention or of a camera as defined in The fourth aspect of the invention.

Preferred macromolecules and glasses Appropriate and preferred are described above.

Mesoporous glasses as defined in relationship with the first and second aspects of the invention have not previously contemplated as nucleants for the macromolecular crystallization. Its usefulness in nucleation can due to the ability of macromolecules to crystallize, find a pore of a suitable size, be retained by pores and, as a result, associate with other macromolecules Similar. The retention of macromolecules in a pore forms a local concentration appropriate for nucleation.

A sixth aspect of the invention provides a kit of parts comprising a crystallization agent and a mesoporous glass as defined in relation to the first or the second aspect of the invention, or a camera as defined in the fifth aspect of the invention.

Storage of glass nucleants mesoporous should preferably be done in the presence of a powder desiccant standard indicator.

With "crystallization agent" we include any one or more of a range of precipitators such as polymers and organic solvents and crystallization agents such as salts. Examples Specific precipitator specific include polyethylene glycol 400, polyethylene glycol 4000, dihydrogen phosphate monosodium and ammonium sulfate.

It should be appreciated that the kits of the invention are appropriate for all crystallization methods, including microlot methods, hanging drop by vapor diffusion, drop posante, sandwich drop and free crystallization by diffusion of surface. It would also be possible to insert the glass nucleant into gelled drops

The kits of the invention may comprise also crystallization plates or sheets and / or filters or chambers of crystallization. When the kit also includes plates crystallization, it is preferable that the plates be plates multiwell, which would be particularly appropriate for a High performance analysis system.

In a preferred embodiment of this aspect, the kit also includes oil to coat the sample of crystallization. Appropriate oils include paraffin such as is available from Hampton Research, CA 92677-3913 USA, catalog number HR3-411.

In an alternative embodiment, the kit can be one appropriate for the scrutiny of the crystallization of macromolecules In this embodiment, the kit may also include one any or more of a range of buffers (which may cover a variety of pH values) and / or any one or more of a range of you go out. Appropriate buffers and salts are well known in the crystallization technique and include, as buffers, Na-HEPES pH 7.5 and tris hydrochloride pH 7.5, and, as salts, 0.2 M ammonium sulfate and 0.2 sodium acetate trihydrate M. Advantageously, the kit may additionally include filters and multiwell crystallization plates.

A seventh aspect of the invention provides an automated method to crystallize a macromolecule consisting of adding a glass to the crystallization test mesoporous as defined in the first and second aspects of the invention using an automated dispensing system.

Appropriate and preferred glasses are those that have described above.

Advantageously, crystallization is part of a selection or optimization for the conditions of crystallization.

Mesoporous glass may be prepared for administer it to the assay through any appropriate means. Preferably, the mesoporous glass is in the form of small fragments of no more than 10 micrometers in any of its dimensions. In a preferred embodiment, the mesoporous glass is Prepare as described above.

It should be noted that mesoporous glass can be added as a single granule, particle or piece or it can add suspended in a liquid. It is difficult to guarantee, or this entails a degree of error so high that it is not acceptable, that in an automated test a single particle of only 10 micrometers in size and no particles are added or well too many. Clearly, therefore, an advantage of the method in that mesoporous glass is added in suspension is that it can be handle mesoporous glass more easily, especially from automatically through an automated delivery system liquid. The formation of a suspension of glass particles mesoporous in a liquid to dispense a specific volume of liquid that includes at least 1 piece or fragment of the glass mesoporous, but not too many pieces or fragments, would facilitate the step of adding the fragment to the crystallization sample.

Hence, in a preferred embodiment of this aspect of the invention, mesoporous glass is dispensed as a suspension by means of an automated dispensing system of liquid.

In the protein crystallization technique, they know automated liquid dispensing systems, and a example is the IMPAX system produced by Douglas Instruments, Hungerford, United Kingdom. In this system through the same nozzle can be dispensed in a single drop of various sample crystallization sample components that come from separate deposits. Properly, the automated system useful in this aspect of the invention is programmable so that in the crystallization drop can be dispensed a few volumes and determined liquid or suspension concentrations.

The crystallization assay may consist of any appropriate method, including microlots (Chayen et al (1990) Appl. Cryst. 23: 297; Chayen et al (1992) J. Crystal Growth 122: 176) and vapor diffusion. The microlot method can be altered as described in D'Arcy et al (1996) J. Crystal Growth 168: 175-180 to use a mixture of silicon and paraffin oil on the crystallization sample. To maintain the level of supersaturation appropriate for the growth of the glass and that does not induce nucleation, it is preferable that the oil to be used is one that does not allow appreciable diffusion of water molecules to the interface between oil and air and, therefore, Do not encourage the concentration of the crystallization sample components. So it is preferable that the oil is a branched paraffin on the C20 + scale and not a silicon fluid (such as a polymer of dimethylsiloxane units).

An eighth aspect of the invention provides a glass obtainable or obtained by the method of the first, the second or the eighth aspect of the invention.

As indicated above, it is likely that the use of mesoporous glass in the crystallization material make the macromolecule that is going to crystallize nuclee within the network of pores of the material, and not on the surface of the mesoporous glass. Consequently, the resulting glass can include a glass mesoporous within the crystal structure itself of the glass produced. This glass may be different from the glasses obtained with alternative means (such as using a surface mineral to induce nucleation), since the nucleant can constitute an integral part of the glass instead of a part peripheral

A further aspect of the invention provides a use for automated liquid dispensing system in the method of the seventh aspect of the invention, in which the glass Mesoporous to be dispensed is in the form of a suspension.

An "automated dispensing system of liquid "includes any automated dispensing system of appropriate liquid that is capable of dispensing a volume of suspension of the porous material that is between 2 nl and 1 ml, per for example, between 2 nl and 50 or 20 nl, for example 5 nl or 10 nl (for low volume / high flow assays) or between 0.05 or 0.1 µL and 1 ml (for tests with larger volumes). Preferably the system is like the one described above.

The fiber, mesh or film of the invention It can also be used in an automated system.

The invention will now be explained more. in detail with the help of the following Figures and Examples no limiting

They are incorporated here by reference the documents referred to.

Figure 1: Glasses of alpha-curstacyanine (lobster shell) cultured bioactive vitreous gel particles CaO-P2 O5 {SiO2}. The glasses measure approx. 140 x 80 x 40 µm.

Figure 2: N2 absorption isotherms for 58S (mesoporous) and 45S5 (control) glass powders.

Figure 3: Pore size distributions of 58S and 45S5 glasses.

Figure 4: Schematic diagram of a section cross section of the mesoporous (non-nucleating) glass that has a Narrow distribution of pore sizes.

Figure 5: Schematic diagram of a glass nucleating mesoporous with a wide distribution of diameters of pore, from 7 to 40 nm.

Example one

Method to crystallize crustacyanine using mesoporous glass CaO-P 2 O 5 {SiO 2 as nucleating

The materials tested as nucleating were bioactive vitreous gel particles CaO-P2 O5 {SiO2}. One (58S) presents a distribution of pores as described in the Example 2, and the other (45S5; control) has no detectable pores, as described in Example 2.

Mesoporous nucleating glass CaO-P2O5 -SiO2 (58S) was prepared by hydrolyzing teraethylorthosilicate (TEOS) with H2O in the presence of HNO3 as catalyst. The precursor of P2O5 was triethoxyl orthophosphate (PET) and the precursor to CaO was Ca (NO3) 2.4H2 O. The gel was dried at 60 ° C and 130 ° C and stabilized at 100 ° C to remove water from the pores and the surfaces of the pores.

The 45S5 bioactive glass was prepared by melting SiO 2, P 2 O 5, CaCO 3 and Na 2 CO 3 in a platinum crucible at 1350 ° C and tempering molten glass with water to make a frit that is then crushed to form the non-porous particles

The protein used (alpha-crustalcyanine) at night tends to produce rain of cluttered glass. Yes, tested using nucleants vitreous, larger unit glasses could be obtained in low supersaturation conditions, which would produce if not clear drops

Two sets of drops of control: (1) Under supersaturation conditions that produce glass rain (2) Under conditions of low supersaturation (i.e. lower concentration of precipitant or what is called metastable conditions).

Other drops of the same protein preparation were put in the same conditions as (2) but a set of drops contained mesoporous glass CaO-P2 O5 {SiO2} (added to the drop in 1 or 2 granules), while the other set of drops had not been added glass granules mesoporous The control (1), as expected, produced rains of Glasses during the night. The control (2) remained clear indefinitely

Drops containing the porous nucleant they produced large individual glasses after 2-3 days Figure 1.

The drops that contained the bioactive glass (not porous) 45S5 were kept clear as if they had not been nucleating added.

The methods used were steam diffusion and microlots with 2 µl drops in each case. Rehearsals are incubated at 10 and 4 ° C. The supersaturation conditions they gave place to the glasses at night were:

protein 20 mg / ml 15% PEG monomethyl ether 0.1 M MES buffer pH 6.5 and chloride 10 mM zinc

The metastable conditions under which inserted the nucleants were:

protein 20 mg / ml 12.5% PEG monomethyl ether 0.1 M MES buffer pH 6.5 and chloride 10 mM zinc

Glass characterization methods N 2 adsorption

The adsorption of gas executed using N2 gas in a six-port Autosorb AS6B-KR Quantachrome gas absorption system. The instrument determined the isotherms volumetrically by means of a discontinuous static method at 77 K (Quantachrome N2 Adsorption manual. Autosorb 6®, Quantachrome Corp. USA, 1997). N2 with a transverse area of 0.162 nm2 was used as adsorptive gas (Lowell and Shields Powder Surface Area and Porosity 1984).

Other details are provided in the Example 2.

Example 2

Measurement of pore characteristics N 2 adsorption

The absorption of gas executed using gas N2 in an Autosorb AS6B-KR gas absorption system Quantachrome six ports. The instrument determined the volumetric isotherms by means of a static method discontinuous at 77.4 K [1]. As adsorptive gas, N 2 was used with a transverse molecular area of 0.162 nm2 [2].

Before the adsorption of N 2, all samples were degassed (i.e. exposing the monoliths to a vacuum pressure less than 1 Pa at elevated temperature) at 100 ° C overnight (approx. 16-20 hours). This is made to remove adsorbed gases from the sample surfaces physically, in particular, water vapor [2]. The areas of specific surface areas were determined with respect to the masses of Degassed samples.

Each isotherm comprised a minimum of 20 adsorption and 20 desorption points measured in equilibrium. For the BET surface area calculation was used at least 4 points of adsorption on the relative pressure scale 0.05 <P / P 0 <0.30 (where P_ {0} is the saturated vapor pressure) [3] [4]. Be verified in each case that the slope and the orderly one at the origin of the regression line of the BET points were positive and that the correlation coefficients of linear regression were not less than 0.99991 [1]. Three isotherms of each composition were obtained for ensure that the data were representative. From the amount of nitrogen taken by the sample on a scale of relative pressure of 0.994 <P / P_0 <0.999 could be calculated  the specific pore volume [1]. The size distributions of pore were calculated using the BJH method with the desorption data [5].

Results - Adsorption of N2 Isotherms

The isotherms shown in Figure 2 are the which were obtained for the 45S5 fusion derivative glass and for the sol-gel glass powders. The glass derived from fusion showed a very reduced adsorption, mainly formation in a single layer followed by saturation, revealing some levels insignificant porosity. On the contrary, the isotherm of the 58S sample corresponds to Type IV of the IUPAC classification, which indicates that porous adsorbents have pores with a radius in the 20 to 500 Å scale (diameter on the 4 to 100 nm scale) (i.e. the samples are mesoporoas) [3]. Hysteresis in the area multilayer of the isotherm is associated with capillary condensation of N_ {2} during the filling and emptying of the mesopores of the structure. The hysteresis loop observed here is of type H1 [3]. This indicates that the analyzed materials contained pores open cylindrical

Figure 3 shows the distribution of Pore size in the vitreous gels, calculated using the BJH model [5] in the desorption branch of the isotherm N2.

Summary

one

References

[one]. Manual for Quantachrome N_ {2} Adsorption. Autosorb 6®, Quantachrome Corp. USA, 1997 .

[2]. Lowell S, Shields JE. Powder Surface Area and Porosity. Chapman and Hall 1984 .

[3]. Sing KSW, Everett DH, Haul RAW. Pure and Appl. Chem 1985 ; 57: 603-619.

[4]. Brunauer S, Deming LS, Deming WE, Teller E. Jof Am. Chem. Soc . 1940 ; 62: 1723-1732.

[5]. Barrett EP, Joyney LG, Halenda PP. J of Am. Chem. Soc . 1951 ; 73: 373-380.

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References cited in the description

This list of references cited by the applicant is solely for the convenience of the reader. It is not part of the European Patent document. Although great care has been taken in the compilation of references, errors or omissions cannot be excluded and the EPO rejects any responsibility in this regard .

Patent documents cited in the description

US 4171544 A

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Claims (23)

1. A method to facilitate the crystallization of a macromolecule comprising the step of adding a glass mesoporous to a crystallization sample in which the glass mesoporous comprises pores with diameters between 4 nm and 100 nm and It has a surface area of at least 50 m2 / g. 2. A method to facilitate the crystallization of a macromolecule comprising the step of adding to the sample of crystallization a mesoporous glass of the composition SiO2; CaO-P 2 O 5 -SiO 2 or Na 2 O-CaO-P 2 O 5 -SiO 2, where each of the atoms of Ca, P, Si or Na of the compositions can be substituted by an appropriate atom chosen from B, Al, Ti, Mg or K, and, optionally, the composition may include also heavy elements such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta u another atom with an atomic number greater than 20 to enhance the X-ray diffraction contrast. 3. A method according to Claim 2 wherein the mesoporous glass is of the composition SiO2; CaO-P 2 O 5 -SiO 2 or Na 2 O-CaO-P 2 O 5 -SiO 2. 4. A method according to claims 2 or 3 in which mesoporous glass comprises pores with diameters between 2 nm and 200 nm. 5. A method according to claim 4 wherein the diameter of the pores has a standard deviation of at least 10 nm 6. A method according to any of the previous claims wherein the mesoporous glass has interconnected pores that intersect the surface of the glass. 7. A method according to any one of the preceding claims wherein the crystallization of the macromolecule is induced at a critical level of supersaturation less than that achieved when mesoporous glass is not added to the sample. 8. A method according to any of the Claims 1 to 7 comprising the step of fracturing the mesoporous glass in pieces of submillimeter dimensions before add the mesoporous glass to the crystallization sample. 9. A method according to claim 8 wherein the pieces do not measure more than 200 micrometers in any of their dimensions. 10. A method according to Claim 9 in the that the pieces do not measure more than 100 micrometers in any of their dimensions. 11. A method according to any one of the Claims 8 to 10 in which the fracture is made by cutting with a scalpel or by mechanical means (diamond cutter), or well breaking a large piece into small pieces using some Tweezers or crushing with a small load. 12. A method to determine the structure of a macromolecule that comprises the steps of:

(one)

crystallize the macromolecule in presence of a mesoporous glass as defined in the Claim 1 or 2; Y

(2)

analyze the crystal structure of glass produced in step (1).

13. An appropriate chamber to crystallize a macromolecule, or a fiber, film or mesh, in which said chamber, fiber, film or mesh comprises a mesoporous glass such as defined in relation to claims 1 or 2. 14. A camera, fiber, film or mesh according to the Claim 13 wherein the mesoporous glass forms a coating on the camera, fiber, film or mesh. 15. Use of a mesoporous glass as defined in relation to Claim 1 or 2, or of a chamber, fiber, film or mesh according to claims 13 or 14 in the crystallization of a macromolecule. 16. A kit of parts comprising an agent of crystallization and a mesoporous glass as defined in relation with claims 1 or 2 or a camera as defined in the Claim 13 or 14. 17. An automated method to crystallize a macromolecule comprising the addition of a mesoporous glass as is defined in relation to claims 1 or 2 to a test of crystallization using an automated dispensing system. 18. A method according to Claim 17 in the that crystallization is done in a selection or optimization. 19. A method according to Claim 17 or 18 in which mesoporous glass is added as a suspension in a liquid. 20. A method according to claims 1 to 3 or 12 or from 17 to 19, or a use according to Claim 13, or either a kit according to any one of Claims 14 to 16 in which the mesoporous glass as defined in relation to the Claims 1 or 2 is prepared according to the method of a any one of Claims 8 to 11. 21. A method according to claims 1 to 3 or 12 or from 17 to 19, or a use according to Claim 13, in that the macromolecule is a biological macromolecule. 22. A method or use according to Claim 21 in which the macromolecule is a protein. 23. Use of an automated system of liquid dispensing to dispense a porous mesoporous glass according to the method of claim 19.